绵羊BMP15基因B2突变可视化检测技术的建立

doi:10.16431/j.cnki.1671-7236.2021.03.004

Abstract

Abstract: Bone morphogenetic protein 15 (bone morphogenetic protein 15,BMP15) gene mutation had a great impact on sheep ovulation rate,litter size and fecundity.Therefore,this study was aimed to establish a rapid visual detection technology for B2 mutation of BMP15 gene.Combine the improved amplification refractory mutation system (ARMS) with the nucleic acid dye SYBR Green Ⅰ,and designed ARMS-specific primers for the B2 mutation of BMP15 gene,made the last base at the 3'end of the primer as A,meanwhile designed additional mismatches at the third base of the 3'end of the downstream primer to improve primer specificity.After ARMS PCR,added SYBR Green Ⅰ to the product,and detected the B2 mutation of BMP15 gene by visually observing the color change of the PCR tube.It was found through sequencing that the collected samples were all wild-type.Therefore,the B2 mutation of BMP15 gene template was constructed by overlap extension PCR.The sequencing results showed the B2 mutation of BMP15 gene template was successfully constructed.ARMS PCR results showed that ARMS-specific primers could only amplify mutant templates,could not amplify wild-type templates,and the amplification effect was good.After ARMS PCR,SYBR Green Ⅰ was added and it was found that the known wild-type template was consistent with the negative control and showed the original color of SYBR Green Ⅰ orange-yellow,while the known mutant template was bright green with obvious color changes.Using the established visual detection technology to detect 50 Small-tailed Han sheep genomic DNA (20 samples were added with the constructed B2 mutant template of BMP15 gene),and compare the number of the mutant sample detected visually with the number recorded when the sample was mixed.The results showed that this technology could accurately detect the B2 mutation of BMP15 gene,and the accuracy was as high as 100%.The constructed BMP15 gene B2 mutant template was serially diluted to detect the sensitivity of the visual detection system in this experiment.It was found that the sensitivity of the ARMS visual detection technology reached 0.006 ng/μL.Therefore,the technology established in this study could visually detect the B2 mutation of BMP15 gene in sheep with high accuracy,and it was expected to provide technical support for the breeding of high-fertility sheep.

Key words: sheep; BMP15 gene; B2 mutant; visual detection; ARMS; SYBR Green Ⅰ

CLC Number:

S826

HU Ruirui, LIU Li, GUO Tao, LI Yaxin, WANG Xiaokui, HU Shengwei. Establishment of a Visual Detection Technique for B2 Mutation of BMP15 Gene in Sheep[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(3): 810-817.

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Establishment of a Visual Detection Technique for B2 Mutation of BMP15 Gene in Sheep

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